The present invention relates to the staining of chromosomal material, and more particularly to the formation of G-Bands in plant chromosomes.
Staining of chromatin for microscopic examination has long been used as a method for monitoring chromosomal changes or abnormalities in both plants and animals. Several techniques are available, each of which results in a specific pattern of bands of color deposition in the chromosomes. Among the best known banding techniques are those which induce the formation of C-Bands, G-Bands, and R-Bands. The term "C-Bands" is used to describe the light and dark bands which appear on the chromosomes when viewed through a microscope which are the result of treatment of the cells with certain known staining processes. The locations of these light and dark bands are specific for each specie from which the cells are taken.
G-Bands are much finer than C-Bands, have specific widths, run longitudinally along the chromosomes and have specific intervals between bands. R-Bands are the reverse of G-Bands: dark G-Bands appear as light R-Bands and light G-Bands appear as dark R-Bands.
G-Banding (and its equivalent, R-Banding) is a particularly useful banding technique which was originally described by M. Seabright, Lancet ii, 971 (1971) for which there have been many technical improvements, especially prophase chromosome banding (see J. J. Yunis, 191 Science 1268 (1976)). For instance, G-Bands can be used by medical geneticists to identify cancer and many congenital abnormalities in humans. Although the techniques used to induce G-Bands in chromosomes were not equally successful in all animal species, G-Banding became a widely used technique for the monitoring of chromosomal changes in vertebrate species. If two chromosomes were to exchange segments during cell division (mitosis), C-Banding would not detect the exchange whereas G- or R-Banding techniques can be used to easily monitor such changes. The use of G-Banding has contributed significantly to advancements in human and mammalian cytogenetics.
However, all previous attempts to induce the formation of G-Bands in the chromosomes of plant cells have been unsuccessful, resulting instead in the formation of C-Bands. For instance, K. Taniguchi and R. Tanaka, 9 Scibo 126 (1977) report a procedure for induction of C-Bands and J. K. S. Sachan and R. Tanaka, 51 Jap. J. Genet. 139 (1976), E. J. Ward, 22 Can. J. Genet. Cytol. 61 (1980) and C. Chow and E. N. Larter, 23 Can. J. Genet. Cytol. 255 (1981) all report attempts to induce G-Bands in plant chromosomes which resulted in C-Bands. Additionally, it has been suggested that it is not possible to induce G-Banding in plant chromosomes (see J. Greilhuber, 50 Theor. Appl. Genet. 121 (1977).
In general, currently available banding techniques for staining of both plant and animal cells utilize post-fixation treatments. In accordance with such techniques, the cells are first fixed by interrupting the dynamic processes of the cell as quickly as possible and stabilizing the chemical structures of the cell with a minimum of change. Following fixation, slides are prepared for viewing through the microscope. A variety of chemicals such as urea, potassium permanganate and trypsin may be used to induce post-fixation G-Band formation in air-dried cytological preparations at plant cells.
G-Band formation can also be induced by treating growing cells with chemicals, such as actinomycin D (AMD) which binds with the guanine residues of bihelical DNA (D. Schweizer, 102 Exp. Cell Res. 408 (1978)), before fixation. Attention is called to an article by T. C. Hsu, S. Pathak and D. A. Shafer (78 Exp. Cell Res. 484 (1973)) which discloses the use of AMD, ethidium bromide, Nogalamycin and azure B to induce G- or R-Bands in mammalian chromosomes by prefixation treatment. In general, R-Bands were induced by the same procedure used to induce G-Band formation with the additional post-fixation step of heating the cells in a phosphate buffer solution at 90.degree. C. Although AMD gave excellent results, this technique was not as popular as post-fixation treatments because AMD is highly toxic to living cells. As a result of the high toxicity, when cell cultures are treated with AMD for a few hours, they yield very few actively dividing cells for subsequent staining upon harvest. Treatment of the actively dividing cells with colchicine, or similar agents, creates the potential for a high yield of cells in early mitotic phases because colchicine arrests the mitotic process prior to cell division. Because colchicine treatment makes available a large number of cells that are in the process of dividing, the fact that treatment with AMD is toxic to the cells has little effect on the number of cells available for subsequent pre-fixation treatment to induce G-Bands.
This invention overcomes many of the limitations discussed above and provides, for the first time, a process for the staining of the chromosomes of plant cells which results in the formation of G-Bands.
Using the method of the present invention, plant chromosomes may be banded in patterns which are characteristic for each plant specie. These characteristic banding patterns are of considerable significance to plant breeders and geneticists who monitor the chromosomal complement of the plant specie, for instance, in hybridization studies. In particular, the banding patterns allow the identification of chromosomes and chromosome segments as they might have been exchanged or distributed upon segregation of the chromosomes during mitosis or meiosis.